S100(, a 91 residue protein present in the brain, promotes neurite extension and enhances neuronal survival. Elevated S100( expression is documented for patients with Alzheimer's disease, Down's syndrome, and AIDS-related dementia. Several studies also show that S100( undergoes distinct conformational changes upon binding Ca(II) or Zn(II). To further characterize this important protein by NMR, high yields of S100( were produced from an overexpression plasmid permitting 13C and/or 15N labeling. Thus, 2D NOESY, TOCSY, DQF-COSY, ROESY, 1H-15N HSQC, and 3D 13C-edited NOESY-HSQC, 15N-edited NOESY-HSQC, 15N-edited HOHAHA-HSQC, 15N,15N-edited HMQC-NOESY-HMQC, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, C(CO)NH, H(CCO)NH, and 4D 13C,15N-separated NOESY experiments were collected with either unlabeled, 15N-labeled, or 13C,15N-labeled S100( in the absence of metal ion. These NMR experiments permitted the total sequential assignment of the backbone and side-chain resonances of S100(, and the secondary structure was determined based on the resonance assignments, chemical shift data, NOE data, and amide exchange rate data. The secondary structure of S100( as determined by NMR contains four helices separated by four loops, which is consistent with the prediction that S100( contains two helix-loop-helix Ca(II)-binding motifs known as "EF-hands."